The following steps were used to purify bovine enterokinase from duodenal mucosa: a) extraction with deoxycholate, b) salt fractionation, c) DEAE-cellulose chromatography, and d) affinity chromatography on Sepharose-bound pancreatic trypsin inhibitor. The product was homogeneous on disc gel electrophoresis and SDS-gel electrophoresis. The molecular weight of the intact protein is about 150,000. The enzyme contains a heavy chain with a molecular weight of 110,000 and a light chain, a catalytic subunit, of molecular weight of 35,000. One or more disulfides link the chains. The amino acid composition of bovine enterokinase resembles that of the porcine enzyme, reported from another laboratory. The composition of the light chain is remarkably similar to bovine trypsin. The catalytic constants toward trypsinogen and synthetic peptide substrates were obtained and resemble those of porcine enterokinase. We found the specificity equal with lysine and arginine substrates. The protein proteinase inhibitor, pancreatic trypsin inhibitor, has an appreciable association constant for bovine enterokinase while other inhibitors interacted weakly or not at all.